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[43] Preparation of radiolabeled ceramides and phosphosphingolipids
[43]
RADIOLABELED
CERAMIDES
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[43] P r e p a r a t i o n o f R a d i o l a b e l e d C e r a m i d e s and Phosphosphingolipids By ALICJA B1ELAWSKAand YUSUF A. HANNUN Introduction Radiolabeled sphingolipids (radio-SLs) with high specific activity, labeled at designated parts of the molecule, allow thorough investigation of their metabolic fate and quantitation of enzyme activities in sphingolipid metabolic pathways. To prepare structurally homogeneous radio-SLs, welldesigned synthetic approaches and precursors should be employed, which are available only in the course of stereospecific synthesis. In general, a few methods can be considered for the preparation of radiolabeled phospho-SLs: (1) biosynthetic approach using radioactive precursors such as L-serine, fatty acids, choline, or 32p. This approach generally results in low activity and leads to mixtures. (2) Labeling in the course of total synthesis following established synthetic methods. (3) Synthetic or enzymatic approaches that are mainly limited to the transformation of existing functional groups present in the structure of natural and synthetic phospho-SLs. This latter approach was commonly used during the early stage of sphingolipid biochemistry and, due to its simplicity and effectiveness, still remains a "method of choice" for many radiolabeled analogs. Homogeneous simple synthetic radio-SLs are available at the level of sphingosine and ceramide stereoisomers. Unfortunately, to date, full access to structurally homogenic complex radio-SLs is limited because of deficiency of the suitable synthetic precursors. Radioactive complex SLs are mainly reconstructed from their natural forms. We would like to stress two general problems encountered with the utilization of natural SLs as precursors in the radiolabeled synthesis. First, naturally occurring SLs exist as nonhomogeneous mixtures due to variation in the length, saturation, and hydroxylation of the sphingoid backbone and the N-acyl groups. Second, the commonly applied procedures for the isolation and preparation of simple SLs from complex precursors (such as lysosphingomyelin from sphingomyelin) can lead to a mixture of erythro- and threo-diastereoisomers due to epimerization at the C-3 position, a result of the tendency of the allylic alcohol to racemize during acidic hydrolysis. This article presents synthetic and enzymatic methods for the preparai K. A. Karlsson,
Chem. Phys. Lipids
METHODS IN ENZYMOLOGY, VOL, 311
5, 6 ( 1 9 7 0 ) .
Copyright © 1999 by Academic Press All rights of reproduction in any lorm reserved. 0076-6879/99 $30.00
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tion of ceramide, SM, and ceramide phosphate labeled in the N-acyl group; SM and lyso-SM labeled in the choline group; ceramide phosphate and sphingosine phosphate labeled in the phosphate group; and the combination thereof leading to multiple-labeled analogs. Materials [3H]Sodium borohydride (450 mCi/mmol), [3H]acetic anhydride (85 mCi/mmol), [1-14C]hexanoic acid (55 mCi/mmol), N-hydroxysuccinimidyl ester of [1-14C]hexanoic acid (50 mCi/mmol), [9,10-3H]palmitic acid (50 Ci/ mmol), [1J4C]palmitic acid (54.2 Ci/mmol), [14C]methyl iodide (55.7 mCi/ mmol), and [3H]methyl iodide (82 Ci/mmol) are obtained from American Radiolabeled Chemicals (St. Louis, MO). Trifluoroacetic acid (TFA) and phytosphingosine hydrochloride are from Sigma. Amberlite IRA 400 (OH-) resin and palmitic acid are from Fluka Chem. Corp. (Milwaukee, WI). CH3COONa × 3H20, cyclohexylamine, diisopropylethylamine, Na2S203, and diethylphosphoryl cyanide (DEPC) are from Aldrich Chemical Company, Inc. (Milwaukee, WI). TLC plates: The following TLC plates are used as indicated in the specific sections: (1) TLC-1; precoated TLC sheets of silica gel 60 F 254 (0.2 layer thickness) are from EM Separation Technology, (2) TLC-2; precoated thin-layer plates Kieselgel 60 (0.25 mm layer thickness) are from EM Separation Technology, (3) TLC-3; precoated thin-layer plates PK5 silica gel 150 A (0.5 mm layer thickness) are from Whatman Laboratory Division, and (4) TLC-4; Linear-K preadsorbent strip, silica gel 150A (1.0 mm layer thickness) are from Whatman Laboratory Division. Silica gel 60 (particle size 0.040-0.063 mm) (230-400 Mesh ASTM) for column chromatography CAS 63231-67-4 is from EM Separation Technology. Sep-Pac Plus Silica, Sep-Pac Plus CM, and Sep-Pac Plus C18 cartridges are from Millipore Corporation, Waters Chromatography Division (Milford, MA). All solvents are of analytical grade or better and are obtained from Mallinckrodt, Burdick and Jackson, or J. T. Baker. Anhydrous N, N-dimethylformamide: DMF (packaged in prescored ampoules) is from Aldrich Chemical Company, Inc. General Methods Used during Synthesis Tetrahydrofuran (THF) is freed of peroxides by passing over basic alumina, followed by saturation with dry argon. Cyclohexylamine, diisopropylethylamiue, and diethylphosphoryl cyanide are distilled before using.
[43]
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Key Precursors for Preparation of Radiolabeled Sphingolipids and Analytical Standards Stereoisomers of sphingosine, sphinganine, and their N-tert-butoxycarbonyl and N-acyl derivatives, N-hydroxysuccinimidyl ester of [1-UC]hexa noic acid, ceramide-l-phosphoryl-N,N-dimethylethanolamine, and sphingosylphosphorylamine are synthesized according to the procedures presented elsewhere in this volume. 2 Preparation of stereoisomers of [3-3H]sphingosine and [3-3H]ceramide is presented elsewhere in this volume. 3
Thin-Layer Chromatography Purity of the compounds, the reaction progress, and chromatographic elution profiles are checked routinely by thin-layer chromatography using analytical TLC plate: TLC-1. Purification is performed using preparative TLC plates: TLC-2, -3, or -4.
Flash Column Chromatography Separation of reaction products and purification of the obtained compounds are achieved by flash chromatography under positive pressure of nitrogen gas as described previously. 4 Fractions containing impure material are rechromatographed.
Solvent Systems The solvent systems used for TLC and/or flash column chromatography purification are (A) chloroform-methanol-2 N ammonium hydroxide (4 : 1 : 0.1, v/v); (B) methylene chloride-methanol (93 : 7, v/v); (C) chloroform-methanol-15 mM CaC12 (60:35:8, v/v); (D) chloroform-methanol (19 : 1, v/v); (E) chloroform-methanol-water (65 : 25 : 4, v/v); (F) chlorof o r m - m e t h a n o l - a m m o n i u m hydroxide-water (160 : 40 : 1 : 3, v/v); (G) chloroform-methanol-concentrated ammonium hydroxide (65 : 35 : 8, v/v); (H) chloroform-methanol-ammonium hydroxide-water (65 : 35 : 2.5 : 2.5, v/v); (I) chloroform-methanol-water (3 : 48 : 47, v/v); (J) chloroform-methanolwater (86 : 14 : 1, v/v); (K) methanol (containing 7% water and 1% concentrated ammonium hydroxide, v/v) and chloroform (1 : 1, v/v); (L) chloroform-methanol-water (60:35:8, v/v); (M) n-butanol-acetic acid-water (3 : 1 : 1, v/v); (N) chloroform-methanol-ammonium hydroxide (60 : 30 : 5, 2 A. Bielawska,Z. Szulc, and Y. A. Hannun, MethodsEnzymol. 311 [44] 1999 (this volume). 3 A. Bielawska,Y. A. Hannun, and Z. Szulc,Methods Enzymol. 311 [42] 1999 (this volume). 4W. C. Still, M. Kahn, and A. Mitra, J. Org. Chem. 43, 2923 (1978).
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v/v); (P) c h l o r o f o r m - a c e t o n e - m e t h a n o l - a c e t i c acid-water (10 : 4 : 3 : 2 : 1, v/v); (R) chloroform-methanol (2:1, v/v); and (S) chloroform-methanol (1:2, v/v). Analytical Assays C o m p o s i t i o n and Diastereoisomeric Purity o f Sphingoid Bases
The purity of sphingosine, sphinganine, and their radiolabeled analogs is evaluated by high-performance liquid chromatography (HPLC) using eicosasphingosine (C20-sphingosine) as an internal standard, as described by Merrill et al. 5 Radioactivity M e a s u r e m e n t
Radioactivity is measured on a Wallac LKB 1214 Rackbeta liquid scintillation counter with High Flash Point Cocktail Safety-Solve (Res. Product Int. Corp. Mount Prospect, IL) as the scintillation fluid. Radioactivity Detection
Detection and quantitation of the radioactivity of the obtained radiolabeled compounds are performed by exposing TLC plates containing radioactive compounds to TLC/Scanner (System 200 Imaging Scanner, Bioscan, Inc. Washington, DC) or to Phosphoimager (Molecular Dynamics, Sunnyvale, CA) Analytical TLC plates containing tritium can be exposed to radiography [after using surface autoradiography enhancer (En3Hance spray, NEN Research Products, Boston, MA]. L i p i d Extractions
Lipid extractions from the crude reaction mixtures are performed using the Bligh and Dyer extraction method 6 or the Folch et al. 7 procedure. Phosphorous measurement is performed using a published method. ~ DG-kinase phosphorylation is performed using a published methodg; this method is also presented in volume 312. l° A. H. Merrill, Jr., E. Wang, R. E. Mullins, W. C. L. Jamison, S. Ninkar, and D. Liotta, Anal Biochem. 171, 373 (1998). 6E. A. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 7j. Folch, M. Lees, and G. H. Sloane-Stanley,J. Biol. Chem. 226, 497 (1957). 8B. N. Ames, Methods' EnzymoL 8, 115 (1966). 9p. p. Van Veldhoven, W. R. Bishop, D. A. Yurivich, and R. M. Bell, Biochem. Mol. Biol. Inter. 36, 21 (1995). 10D. Perry, A. Bielawska, and Y. A. Hannun, Methods' Enzymol. 312 (1999).
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIP1DS
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OH X - C(O)([3HICH2)nCH3
R2OA~'~'~N (CH2)12C H3 NH-C(O)([3H]CH2)nCH3
OH R2OA~"~
(CH2)12CH3 - -
NI-I2
OH X -14C(O)(CH2)nCH3
R2 = H, phosphate, choline phosphate or oligosaccharide chain X = H, C1, active ester group
~ R20
(CH2)12CH3 NH- C(O)(CH2)nCH3
Fl(~. 1. Approach to the synthesis of [3H]- and [14C]sphingolipidslabeled in the N-acyl group.
P r e p a r a t i o n of S p h i n g o l i p i d s L a b e l e d i n t h e A m i d e M o i e t y
Principles C e r a m i d e a n d its m o r e c o m p l e x derivatives are synthesized f r o m the c o r r e s p o n d i n g s p h i n g o i d bases p r e p a r e d synthetically,2 n a t u r a l l y , 1~-16 or from lyso-SLs by classical m e t h o d s used in p e p t i d e chemistry. S t r i n g e n t c o m p e t i t i o n in the reactivity of the a m i n o a n d hydroxyl groups in SLs t o w a r d O- a n d N - a c y l a t i o n favors N - a c y | a t i o n products. T h e a m i d e b o n d is usually p r e p a r e d by the action of acid a n h y d r i d e s 17,18 or acid halides 192° by t r a n s a c y l a t i o n of the activated fatty acids as N - h y d r o x y s u c c i n i m i d e esters, 2L22 or by p - n i t r o p h e n y l e s t e r s Y A l t e r n a t i v e m e t h o d s have b e e n H R. C. Gaver and C. C. Sweeley, J. Am. Oil Chem. Soc. 42, 294 (1965), 12R. Cohen, Y. Barenholtz, S. Gan, and A. Dagan, Chem. Phys. Lipids 35, 371 (1984). 13p. p. Van Veldhoven, R. J. Foglesong, and R. M. Bell, J. Lipid Res. 30, 611 (1989). ~4p. p. Van Veldhoven and G. P. Mannaerts, J. Biol. Chem. 266, 12502 (1991). ~5M. Buneman, K. Liliom, B. T. Brandts, L. Pott, J. L. Tseng, D. M. Desiderio, G. Sun, D. Miller, and G. Tigyi, E M B O J. 15, 101 (1996). ~ A. Kisic, M. Tsuda, R. J. Kulmacz, W. K, Wilson, and G. J. Schroepfer, J. Lipid Res. 36, 787 (1995). 17R. C. Gaver and C. C. Sweeley, J. Am. Chem. Soc. 88, 3643 (1966). ~ A. Bielawska, H. M. Crane, D. Liotta. L. M. Obeid, and Y. A. Hannun, J. BioL Chem. 268, 26226 (1993). 19K. C. Kopaczyk and N. S. Radin, J. Lipid Res. 6, 140 (1965). 2, A. Bielawska, D. Perry, S. Jayadev, C. McKay, J. Shayman, and Y. A. Hannun, J. Biol. Chem. 271, 12642 (1996). 21 y. Lapidot, S. Rapport, and Y. Wolman, J. Lipid Res. 8, 142 (1967). 27A. Futerman and R, E. Pagano, Methods Enzymol. 209, 437 (1986). 2:tB. Neises, T. Andries, and W. Steglich, J. (?hem. Soc. Chem. Commun. 1132, (1982).
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CHEMICALAND ENZYMATICSYNTHESES
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developed using coupling agents such as N-ethoxycarbonyl-2-ethoxy-l,2dihydroquinoline, 24 dicyclohexylcarbodiimide (DCC), 12 diphenyl phosp h o r a z i d a t e y 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,26'27 ethylchIoroformate, 28 pentafluorophenol, 29 or diethylphosphoryl cyanide ( D E P C ) ? ° A new enzymatic method for the preparation of radioactive ceramides using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase (SCDase) has been published? l Four approaches for the radio acylation of the amino group of sphingoid bases employ (1) acetic anhydride, (2) acyl chlorides, (3) N-hydroxy succinimidyl esters, and (4) fatty acids and D E P C as a coupling agent. To synthesize C2-ceramides, use the first approach; to prepare long chain ceramides, use the second approach; and to synthesize C6- and Cs-ceramides, use the last approach. The D E P C method employs diethylphosphoryl cyanide as a coupling agent in the presence of triethylamine and is rapid, free of racemization, utilizes acids without prior derivatization, and generates relatively pure products. We have noticed that this reaction needs to be carried out in anhydrous conditions, under nitrogen atmosphere, and at 0 °. The use of freshly distilled D E P C and triethylamine is critical. Following these rules we have synthesized all stereoisomers of [14C]C6- and [14C]dihydro-C6ceramides with about 90% yield. The acid chloride method (the second approach), which we have named the "one pot" procedure, employs a simple and effective protocol of in situ generation of the acid chlorides from fatty acids and thionyl chloride or oxalyl chloride ~9'2° following the introduction of a THF-aqueous CH3COONa reaction system and selected sphingosines. Using this method, stereoisomers of various radiolabeled long chain ceramide and dihydroceramides can be synthesized with 70-85% yield.
24Z. Dong and J. A. Butcher, Jr., Chem. Phys. Lipids 66, 41 (1993). 25S. Yamada, N. Ikota, T. Shioriand, and S. Tachibana, J. Am. Chem. Soc. 97, 7174 (1975). 26S. Hammarstrom, J. Lipid Res. 12, 760 (1971). 27T. Levade, S. Gatt, A. Maret, and R. Salvayre,J. Biol. Chem. 266, 13519 (1991). 2s D. Aquotti, S. Sonnino, M. Masserine, L. Casella, G. Fronza, and G, Tenamanti, Chem. Phys. Lipids 40, 71 (1986). 29R. Jennemann, C. Gnewuch, S. Bolets, B, L. Bauer, and H. Wiegandt, J. Biochem. 115, 1947 (1994). 3oj. K. Anand, K. K. Sadozai, and S.-I. Hakamori, Lipids 30, 995 (1996). 31S. Mitsutake, K. Kita, N. Okino, and M. Ito, Anal Biochem. 247, 52 (1997).
[43]
RAD1OLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
505
Preparation of Radiolabeled Ceramides
Synthesis of N-[1J H-acetyl]-D-erythro-sphingosine: N-J1J H]D-erythro-CTceramide This section presents the synthesis of stereoisomers of C2-ceramide and C2-dihydroceramide and the preparation of N-acetylphytosphingosine, all labeled in the acetyl group, by a method 18 adapted from the procedure for the preparation of nonradioactive C2-ceramide. 17'j8 [3H]Acetic anhydride (5 mCi, 59 /xmol) is cooled in an ice bath and diluted with 100/xmol (5/xl) of cold acetic anhydride in 5 ml of dry methanol. D-erythro-Sphingosine (12 rag, 40 txmol) is dissolved in 2 ml of dry methanol and is added to the methanol solution of [3H]acetic anhydride. The reaction mixture is stirred in an ice bath for 4 hr, when the TLC-1 solvent shows that the reaction is complete. Cold water (5 ml) is added dropwise to produce a white precipitate, and the mixture is stirred in an ice bath for an additional 30 rain. The precipitate is separated by centrifugation (3000 rpm, 5 min, 5°), separated from the supernatant, dissolved in 5 ml of chloroform, and washed with 1 ml of 0.1 N NaOH. Phases are separated by centrifugation, and the organic phase is washed with water until no radioactivity is present in the upper phase. The organic phase is dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-4 in solvent A. The area corresponding to C2-ceramide is scraped, and ceramide is extracted three times with 5 ml of solvent R. The extraction efficiency is evaluated quantitatively by scintillation counting. Combined extracts are dried under vacuum to give 12.5 mg (91.5% yield) of [3H]I>erythro-C2ceramide (specific activity: 15.75 mCi/mmol). The purity of this product (-96% pure) is established by analytical TLC-1 in solvent B (Rf = 0.21) and in solvent A (Rf = 0.64). This product is dissolved in solvent R at 10 mM and is stored at - 2 0 ° for several months. Using the just-described procedures, the remaining C2-ceramide and C2-dihydroceramide stereoisomers--L-threo-(2S,3S),D-threo-(2R,3R), and L-erythro-(2R,3S)--can be prepared starting from the corresponding sphingosine and sphinganine isomers. [3H]C2-ceramides/dihydroceramides labeled in the sphingosine/sphinganine backbone can be prepared by the acylation of [3-3H]sphingosine and [3-3H] - or [4,5-3H]sphinganines with unlabeled acetic anhydride following the just-described procedure.
Synthesis of N-[1-3H-acetyl]phytosphingosine: N-[1--~H] C2-phytocerarnide [3H]Acetic anhydride (5 mCi, 59 p~mol) is cooled down in an ice bath and diluted with 100/xmol (5 ~1) of cold acetic anhydride in 5 ml of dry methanol. Phytosphinogosine hydrochloride (14.2 mg, 40/~mol) is dissolved
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CHEMICAL AND ENZYMATIC SYNTHESES
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in 2 ml of dry methanol, and the pH is adjusted to 9 with 2 N NaOH in methanol and added to the methanol solution of [3H]acetic anhydride. The reaction mixture is stirred in an ice bath for 4 hr. TLC-1 in solvent A shows that the reaction is complete. The product is isolated and purified following the procedure developed for the C2-ceramide preparation. After final purification, [3H]C2-phytoceramide is obtained in 77% yield (12.2 mg, specific activity: 15.75 mCi/mmol), with a purity of -98% as established by analytical TLC-1 in solvent A ( R f = 0.4).
Synthesis of N-[l YC-Hexanoyl]-D-erythro-sphingosine: N-[1J4C]-D-erythro-C6-ceramide This section presents the synthesis of stereoisomers of C6-ceramide and C6-dihydroceramide by methods that have been adapted from general procedures for the preparation of radioactive ceramides by the DEPC method 3° and by the NHS ester method. 21'22 Via the DEPC Method. I>erythro-Sphingosine (4.9 mg, 16.4/xmol), in freshly distilled and dry DMF-methylene chloride 1:3 mixture (2 ml), is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing [1J4C]hexanoic acid (0.9 mCi, 16.4 txmol) in 900 /xl of dry methylene chloride followed by the addition of 32.8/xmol (11 txl) of freshly distilled DEPC. The volume of the solvents is adjusted to 11 ml, keeping the proportion DMF-methylene chloride as 1 : 3 (v/v). After flushing with argon, the reaction mixture is cooled to 0°, and freshly distilled triethylamine (16.4/xmol, 9/xl) is added under argon. Stirring under argon is continued for 2 h, solvents are evaporated, and the product is extracted by the Bligh and Dyer 6 procedure. The upper phase is reextracted with 2 ml of chloroform; liquid scintillation counting detects almost no radioactivity in the upper phase. TLC analysis of the radioactive organic phase is performed in solvent B (C6-ceramide: Rf = 0.34) and in solvent A (C6-ceramide, R f = 0.74). Solvent A, which separates well ceramides from unreacted fatty acids and sphingosine, shows that -80% of radioactivity is incorporated into ceramide. The organic phase is dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-4 in solvent A. The area corresponding to C6-ceramide is scraped, and ceramide is extracted three times with solvent R. Extraction efficiency is evaluated by liquid scintillation counting. Combined extracts are dried under vacuum to give 5.54 mg (85% theoretical yield) of N-[1-~4C]D-erythro-C6-ceramide. The purity of this product was -95% as established by analytical TLC-1 in solvents C and A. This product is dissolved in solvent R at 10 mM (specific activity: 1.22 × 105 cpm/nmol) and stored at - 2 0 ° for several months.
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Using the just-described procedures, the remaining C6-ceramide and C6-dihydroceramide stereoisomers--L-threo-( 2S,3S), D-threo-( 2R,3 R ), and berythro-(2R,3S) can be prepared starting from the corresponding sphingosine and sphinganine isomers. Via the NHS Ester Method. D-erythro-Sphingosine (6.0 mg, 20/xmol) in 1 ml of dry DMF is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing N-hydroxysuccinimidyl ester of [1-14C]hexa noic acid (1.0 mCi, 20/xmol) in 1 ml of dry DMF followed by the addition of 10/xl of freshly distilled diisopropylethylamine. The reaction is allowed to proceed at room temperature under nitrogen atmosphere in the dark, with stirring. Progress is monitored periodically by TLC-1 in solvent C. After 15 hr, the reaction is almost complete, the reaction mixture is acidified with 20/xl of 3 N HC1, and the mixture is dried completely under a stream of nitrogen. The product is extracted by the Bligh and Dyer 6 procedure. The upper phase is reextracted with 2 ml of chloroform, dried under nitrogen gas, dissolved in solvent R, and purified by preparative TLC-3 in solvent E or F. The area corresponding to C6-ceramide is scraped, and ceramide is extracted three times with 5 ml of solvent R. The extraction efficiency is evaluated by liquid scintillation counting. The combined extracts are dried under vacuum to give 6.4 mg (80% theoretical yield) of N-[1-14C]D-erythroC6-ceramide. The purity of this product is -95% as was established by analytical TLC-1 in solvents C and A.
Synthesis of N-[3H or NC-palrnitoyl]D-erythro-sphingosine: N-[9,10-~H]D erythro-C i6-ceramide or N-[1-14C] D-erythro-C m-ceramide This section presents the synthesis of stereoisomers of C16-ceramide and C16-dihydroceramide via an acyl chloride method that has been adapted from the procedures for the preparation of nonradioactive ceramides and their analogs. ~9'2° [9,10-3H]Palmitic acid (4.8 mCi, 0.08/xmol), diluted with 8/xmol of cold palmitic acid, is refluxed with 0.5 ml of oxalyl chloride in dry benzene for 1 hr under nitrogen atmosphere. 16The solution is then evaporated to dryness under vaccum. D-erythro-Sphingosine (2.6 mg, 8 txmol) dissolved in 2.5 ml of THF is added via syringe to the crude [3H]palmitic chloride followed by the addition of 1.25 ml of 50% CH3COONa. The reaction mixture is stirred vigorously for 4 h. Ceramide is isolated by partitioning with chloroform (6 ml)-methanol (3 ml)-water (2.25 ml). The phases are separated, and the lower phase (8 ml) is washed twice with 2 ml of water. More than 90% of the theoretical radioactivity (calculation from the specific activity of the original [3H]palmitic acid) is recovered in the lower organic phase with almost no radioactivity in the upper phase. TLC-1 analysis of the
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CHEMICAL AND ENZYMATIC SYNTHESES
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organic phase, performed in solvent B (separates ceramides from esterified ceramides) and in solvent A (separates ceramides from unreacted fatty acids and sphingosine), shows that -90% of radioactivity is incorporated into N-[9,10-3H]D-erythro-C16-ceramide. Ceramide is purified by preparative TLC-3 in solvent A, the area corresponding to C16-ceramide is scraped, and ceramide is extracted three times with solvent R. The extraction efficiency is monitored by liquid scintillation counting. The combined extracts are dried under vacuum and repurified with solvent B. The final [3H]Derythro-C16-ceramide (82% yield) is dissolved in solvent R at 10 mM (specific activity: 1.3 × 105 dpm/nmol). The purity is -96%, as established by analytical TLC-1 in solvent B (Rf = 0.21) and in solvent A (Rf = 0.64). This procedure can be used to prepare the remaining C16-ceramide and C~6-dihydroceramide isomers and other Cn-ceramides.
Preparation of Sphingomyelin Labeled in the N-Acyl Group
Synthesis of N-[1-14C-Hexanoyl]-D-erythro-sphingosylphosphorylcholine: N-[1-NC]-C6-SM Sphingomyelin labeled in the N-acyl group is obtained by N-acylation of D-erythro-sphingosylphosphorylcholine via the DEPC method or via the NHS ester method, following procedures developed for N-[1-~4C]ceramide preparation. 26,34 D-erythro-Sphingosylphosphorylcholine (6.0 mg, 13 /xmol) in 2 ml of dry DMF is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing N-hydroxysuccinimidyl ester of [1J4C]hexanoic acid (0.65 mCi, 13/xmol) in 1 ml of dry DMF followed by the addition of 10 /xl of freshly distilled diisopropylethylamine. The reaction is allowed to proceed at room temperature under nitrogen atmosphere in the dark, with stirring. Progress is monitored periodically by TLC-1 in solvent C. After 25 hr the reaction is almost complete, and the reaction mixture is acidified with 20/xl of 3 N HC1 and then dried completely under a stream of nitrogen. The product is extracted by the Bligh and Dyer procedure. The upper phase is reextracted with 2 ml of chloroform, dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-3 in solvent E or F. The area corresponding to C6-SM is scraped, and sphingomyelin is extracted five times with 5 ml of solvent R. The extraction efficiency is evaluated by liquid scintillation counting. The combined extracts are dried under vacuum to give 5.3 mg (75% theoretical yield) of N-[1-14C]D-ervthro-C6-SM.The purity of this product is -95% as established by analytical TLC-1 in solvents C and G.
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R A D I O L A B E L E D C E R A M I D E S A N D PHOSPHOSPH1NGOLIP1DS
509
Preparation of Radiolabeled Sphingomyelin Principles Sphingomyelin can be radiolabeled (Fig. 2) in various parts of the molecule, depending on the experimental needs. Reduction of 3-keto-SM labels the C-3 position of the sphingosine backbone ([3-3H]-SM). N-acylation of sphingosylphosphorylcholine (lyso-SM) with radioactive fatty acids generates SM labeled in the N-acyl group (N-[1J4C-acyl]-labeled SM, see procedure described earlier). The quaternization procedure of ceramidel-phosphoryl-N,N-dimethylethanolamine (demethylated-SM) generates [14C- choline]-labeled SM, and this method is presented next. Chemically homogeneous SMs are available presently in a limited scope as their syntheses were described only for a few selected D-erythro series. 32"33 These multistep methods have not been adapted and optimized yet to the needs of SM radiochemistry. A synthetic approach used by Bitmann's group 33may be considered as a method for the preparation of stereoisomers of radiolabeled SM if radiolabeled ceramide stereoisomers are used as substrates. Also, by employing the [3H]trimethylamine at the last synthetic step, [3H-choline]-SM or double-labeled-SM (in the choline and in the amide part) may be prepared. Radiolabeling in the Choline Moiety. This method was developed for natural SM 34,35 and involves the demethylation of SM to ceramide 1-phosphoryl-N, N-dimethylethanolamine (de-SM) and subsequent quaternization with radioactive [t4C]- or [3H]methyl iodide. Quaternization of de-SM with [3H]methyl iodide allows the preparation of [3H-choline]SM with a high specific activity (~80 Ci/mmol). The demethylation process is presented elsewhere in this volume. 2 Preparation o f [14C-choline]Sphingomyelin
This section presents the synthesis of [14C-choline]SM by a method that has been adapted from the procedure published by Stoffel et aL 34 and the synthesis of [14C-choline]lyso-SM, obtained from [14C-choline]SM by a method that has been adapted from previously described procedures. ~2 15 [~4C]Methyl iodide (5 mCi, 89.8/~mol) is dissolved in 2 ml of dry methanol and transferred to an air-evacuated flask containing 5 ml of milky 3~R. R. Schmidt,in "Synthesisin LipidChemistry"(H. P. Tyman,ed.), p. 93. BrunelUniversity, Uxbridge, UK, 1996. ~3H. S. Byun, R. K. Erukulla, and R. Bittman, J. Org. Chem. 59, 6495 (1994). 34W. Stoffel,D. LeKim,and T. S. Tsung, Hoppe-Seyler's Z. Physiol. Chem. 352, 1058 (1971). 3~W. Stoffel,Methods" Enzymol. 35, 533 (1975).
510
[43]
CHEMICAL AND ENZYMATIC SYNTHESES A
O ®
O II
,
®o
NHCOR 3-keto-SM
I NaB3H4 o ® II (H3C)3N~'/X,O" ~ ' O C)
s
OH
O ~ ][ JH NHCOR
(CH2)12CH 3
[3-3H]-SM
®
o
OH
II
-
(H3C)3N'v'A~"i ' ~ O ~ {=)
(CH2)12CH 3
N1t2 lyso-SM
R14COOH ®
o II
OH --
(H3C)3NV/X.O" f " O ~ (CH2)12CH3 NH14CO" " R N-[14C-acyl]-SM O II
OH -
(H3C)2NV / X - O ~ X O ~ ( C H 2 ) 1 2 C H OH
3
NHCOR
N-demethylated-SM
14CH3J o ~ Oil ---~4 e pII (H3 C ) 3 N x / / " - . X g - [ X O ~ ( C H 2 ) , 2
6
{~)
Ii
CH3
NHCOR [14C-choline]-SM
FlG. 2. (a) Approaches to the preparation of sphingomyelin radiolabeled in various parts of the molecule. (b) Approach to the synthesis of [3H-] or [14C-choline]-labeled sphingomyelin and lysosphingomyelin.
[43]
511
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
B
O II
®
OH .--NHCOR
SM I C6H5SNa DMSO,80°C (~)
~
(H3C)2HNX//X"K)"
OH
~'NO~(CH2)12CH3 NHCOR
N-demethylated-SM
I
I C3H31
14CH3I CH3
O
OH
14 ¢)1 II T-CH3- - ~ x,,/X-,O1 ~ ' O ~ CH3
8
CHo
0
(CH2)12CH3 C Hy----~~ , -
NHCOR
~'--O~(CH2)12C[ 8 NHCOR
CH3
[14C-choline]-SM
[3H-eholine]-SM acid hydrolysis
acid hydrolysis Ct-13 ,~
O
OH
~
II
v
CH3
8
NH2
®1
OH
CH3 ~
H3
O
OFl
~
II
.-=
CH3
8
NH2
®1
[3H-eholine]-Iyso-SM
[14C-choline]-Iyso-SM FIG. 2. (continued)
solution of demethylated sphingomyelin (77.3 rag, 108/.tmol) in dry methanol. This process is repeated four times followed by the addition of freshly distilled cyclohexylamine (11.1 mg, 13 ~1, 112 /,mol) in 200 /,d of dry methanol. This mixture contains more than 80% of the theoretical radioactivity of [14C]methyl iodide. The milky solution becomes progressively clear after - 1 hr and is stirred at room temperature in the dark for 24 hr. TLC1 analysis of the reaction mixture in solvent G shows formation of [14C]SM
t3
512
C H E M I C A L A N D E N Z Y M A T I C SYNTHESES
[43]
(-90% of the total radioactivity), some unidentified minor radioactive byproducts, and the ureacted substrate. Methanol is removed under a stream of nitrogen, and the reaction mixture is treated twice with 3 ml of chloroform (and redried) and dissolved in 10 ml of chloroform. The chloroform solution is washed twice with 5 ml of freshly prepared 5% Na2S203 followed by 2 N HC1 (2 × 4 ml) and water (2 × 4 ml). After centrifugation (3000 rpm, 15 rain, 5°), the upper phase is discarded and the lower phase, with the milky interphase, is diluted with chloroform to get a clear solution (15 ml total) (during the washing process, -15% of radioactivity is lost). The crude product is purified by gradient flash chromatography (11-ram-diameter column) using chloroform-methanol (65:35-5:95, v/v) as an eluting system. Each of the 5-ml fractions is monitored by counting, and elution progress is followed by TLC-1 in solvent G. Unreacted de-SM is eluted with chloroform-methanol (3 : 7, v/v); pure N-[14C-choline]SM elutes with chloroform-methanol (5 : 95, v/v). Fractions containing pure sphingomyelin are combined and evaporated under nitrogen gas to give a white solid product (45.9 rag, 3.5 mCi, 88% yield) (calculated from the specific activity of [14C]methyl iodide). Fractions containing pure de-SM are also combined and evaporated under nitrogen to give a white solid product (22 rag). [HC]Sphingomyelin is dissolved in a mixture of methanol-toluene (1:1, v/v) to yield an activity of 1 × 106 cpm/~l (8.2 mM as determined by phosphorus assay). 12 [~4C]SM can be kept at - 2 0 ° for several months without decomposition.
Preparation of F4C-choline]L ysosphingomyelin N-[~C-choline]Sphingomyelin (0.56 mCi, 6.3 rag, 8.55 /~mol) is hydrolyzed under reflux in 3.5 ml of 1 N HC1 in methanol for several hours with monitoring by TLC-1 in solvent H. After 12 hr, the reaction is complete. Solvent is evaporated and the product is partitioned into the upper phase of the Folch procedure. Briefly, the reaction mixture is redissolved in 2 ml of methanol followed by the addition of 4 ml of chloroform and 1.5 ml of water. Phases are separated and the lower phase (4 ml) is reextracted twice with 3.5 ml of solvent I. The combined upper phases contain more than 90% of [14C-choline]lyso-SM, and sphingosine and N-[~4C]phosphocholine. The pH of the combined upper phases is adjusted to 12-13 with 5 N NaOH and 12 ml of solvent J is added to transfer lyso-SM and sphingosine to the lower phase. This process is repeated three times, with the extraction efficiency evaluated by liquid scintillation counting. The lower phases are combined and evaporated in vacuo. The crude product is purified by preparative TLC-3 in solvent G. The area corresponding to N-[14C-choline]lysoSM is scraped from the plate, and the product is extracted several times
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
513
with 3 ml of solvent K. 15 Extracts containing lyso-SM are combined and evaporated under nitrogen to give a white solid product (2.5 mg, 65% yield, 0.36 mCi). Purity is assessed by TLC-1 in solvents H, L, o r N . 14
Preparation of Radiolabeled Ceramide- 1-Phosphate and Sphingosine- 1-phosphate Principles Synthesis of this class of compounds is difficult to perform, particularly for sphingosine-l-phosphate. The only practical approaches described so far for the preparation of these radiolabeled compounds are enzymatic methods. Enzymatic Methods for the Preparation of [32p, 3H or 14C]Ceramide-1phosphate and [32p or aH]Sphingosine-l-phosphate An enzymatic method employing E. coli diacylglycerol kinase (DGkinase) can be applied for the preparation of radioactive ceramide-l-phosphate (Fig. 3a) and sphingosine-l-phosphate (Fig. 3b). DG-Kinase phosphorylates diacyl and monoacyl glycerols using adenosine triphosphate (ATP) as a phosphate source. 36 This enzyme can also phosphorylate the primary hydroxyl group of ceramide9'13.20,37and their analogs. 3s This method was successfully adopted for the determination and quantitation of the endogenous ceramide in cells and tissue as well as for the fast preparation of a variety of [32p]_, [3HI' or [14C]-labeled ceramide-l-phosphates in moderate yield (-70%). DG-kinase phosphorylates only ceramides posessing the 2S configuration at the C-2 position of sphingosine backbone and generates only (2S,3R) and (2S,3S) stereoisomers. 38 We find that DG-kinase also phosphorylates N-Boc-protected Derythro-and L-threo-sphingosines and sphinganines. Based on that observation and a simple protocol for N-Boc-deprotection [mild acidic, free aminolipid liberation protocol: TFA, - 0 °, Amberlite IRA 400 (OH-), MeOH], we have successfully prepared [32p]_ o r [3H]-labeled sphingosine1-phosphate and sphinganine-l-phosphate without contamination of one stereoisomer by another? 8 The acidic hydrolysis approach, applied pre~(' J. Preiss, C. R. Loomis, W. R. Bishop, R. Stein, J. E. Niedel, and R. M. Bell, J. Biol. Chem. 261, 8597 (1986). 37 K. A. Dressler and R. N. Kolesnik, J. Biol. Chem. 265, 14917 (1990). 3~ A. Bielawska. unpublished observations.
514
[43]
CHEMICAL AND ENZYMATIC SYNTHESES
A OH HO~(CH2)12CH
3 [32p]ATP, DC-kinas~
NHCO (CH2)nCH3
NHCO (CH2)nCH 3
Ceramide
[ 32p]
OH = ATP, DG-kinase H
O
~
~
(CH2)12CH3
O II
OH =
HO ] O ~ ( C H 2 ) 1 2 C H 3 OH ~I_ll4co (CH2)nCH3
NH ' CO (CH2)nCH 3
[14C]Ceramide B
Ceramide-l-phosphate
[14C]Ceramide-1-phosphate
OH
OH
HO~(CH2)12CH3 NHBoc
N-Boc-sphingosine
HO~(CH2)
[32p] ATP, DG-kinase O HO ;HO"
O
ATP, DG-kinase O
OH
32 II
OH
II
-=
12CH3
NHBoc [ 1-3H-or 3-3H] N-Boc-Sphingosine
"(CH2)12CH3
..Px ~ ...@..~'.. HO O]HO" *" "~" ~ "(CH2)I2CH 3 NHBoc
TFA, ~0oC, ] AmberliteIRA 400 (OH-), MeOH " OH
TFA, ~0oC, [ AmberliteIRA 400 (OH-), MeOH - OH
I "~ NHBoc
~
32 II .P-. A J'-.~'-. HO ]OHO" " ~ v -(CH2)12CH3 NH 2 [ 32p] Sphingosine-l-phosphate
{
O
II
HO ] O ' ~ " ~ ' ~ ( C H 2 ) 1 2 C H 3 OH
NH 2
[1-3H - or 3-3H]
Sphingosine-1-phosphate
FIG. 3. (a) Enzymatic method for the preparation of ceramide-l-phosphate III labeled in the phosphate group or in the ceramide portion. (b) Enzymatic method for the preparation of sphingosine-l-phosphate VB labeled in the phosphate group or in the sphingosine portion. Asterisk indicates tritium labeling. (c) Enzymatic method for the preparation of ceramide-1phosphate III B and sphingosine-l-phosphate VB.
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPH1NGOLIPIDS
515
G ® (CH3)3N ~
O II --P'-. O I O1~
OH = " ..~ ~X~-*~X(CH2)12CH3 NH14COR IN-14C-acyl]-SM PL-D oH
HO ] O ' ~ ' ~ " ~ ( C H i ) l i C H 3 OH ~H14COR
[14C]-Ceramide-l-phosphate
® (CH3)3N~
0
OH
II
-
0/ ~ } N O ~ (CH2)12CI-I3 0 • 3H @ NH 2 [3-3H]-Iyso-SM PL-D
I~
I
_OH
HO I o H O ~ ( C H 2 ) 1 2 C H 3 NH 2
[3-3H]-Sphingosine- 1-phosphate F~G. 3. (continued)
viously for the generation of sphingosine-l-phosphate from ceramide phosphates, afforded sphingosine-l-phosphate as a diastereoisomeric mixture. 13,37,38 Other enzymatic methods employ bacterial phospholipase D treatment of SM or lyso-SM 13,14(Fig. 3c) or sphingosine kinase 39,4° for the preparation of radioladeled ceramide- and sphingosine-l-phosphates.
39 W. Stoffel, G. Assmann, and E. Binczek, Hoppe-Seyler's Z. Physiol. Chem. 351, 635 (1970). 4o N. Mazurek, T. Megidish, S.-I Hakamori, and Y. Igarashi, Biochern. Biophys. Res. Commun. 198, 1 (1994).
516
CHEMICAL AND E N Z Y M A T I C SYNTHESES
[43]
Preparation of N-FH-palmitoyl]o-erythro-Sphingosine-l-phosphate: FH]-D-erythro-Clo-ceramide-l-phosphate This section presents an enzymatic method for the preparation of D-
erythro-C16-ceramide phosphate and D-erythro-sphingosine-l-phosphate (labeled with 32p, 14C, or 3H) by DG-kinase action. This method has been adopted from the general method for ATP-phosphorylation of diacylglycerols and ceramides by DG-kinase. 9'36 N-[gH]Palmitoyl-D-erythro-sphingosine(0.2 mCi, 200 nmol) is sonicated for 5 min with 100/zl of 3.75% (w/v) octylglucoside-12.5 mM dioleoylphosphatidylglycerol, made up in 1 mM DTPA, followed by the addition of 350 btl of the reaction mixture containing 120 mM HEPES buffer, pH 7.0-100 mM LiC1-25 mM MgC12-2 mM E G T A - 2 mM dithiothreitol, and 25/.tl of DG-kinase (7 mg/ml). After 10 min, the reaction is started by adding 50 /xl of 10 mM ATP in 20 mM imidazole buffer, pH 6.6-1 mM DTPA. After vigorous mixing, the reaction mixture is left at room temperature for 1 hr, 3 ml of solvent S is added, and the reaction mixture is mixed vigorously for 1 min. Bligh and Dyer extraction is continued by adding 350/zl of 1% perchloric acid followed by 1 ml of chloroform and 1 ml of 1% perchloric acid. Phases are separated by centrifugation (3000 rpm, 5 rain), and the lower phase is transferred to a new vial. This organic phase is dried under nitrogen and subjected to the base cleavage in 2 ml of 0.1 N NaOH in methanol for 30 min, at room temperature. The product is extracted by the Bligh and Dyer method. The lower phase is concentrated under nitrogen gas, and the product is purified by TLC-3 in solvent P and detected using a phosphoimager. The area corresponding to [3H]D-erythro-C16-ceramide1-phosphate (Rf = 0.53) is scraped and extracted five times with 3 ml of chloroform-methanol (l :1, v/v) and is evaluated quantitatively by liquid scintillation counting. The combined exstracts are dried under vacuum to give the final product with -55% yield (calculation is made from the specific activity of the original [3H]D-erythro-C16ceramide). The product is characterized by TLC-1 in solvents P and M and is found to be 95% pure. Following this procedure, other D-erythro- and L-threo-ceramide phosphates and dihydroceramide phosphates can be prepared. However, the phosphorylation efficiency for short chain ceramides is much lower (--35% for C6-ceramide and only -15% for C2-ceramide). 4°
Preparation of o-erythro-F2p]Sphingosine-l-phosphate D-erythro-N-Boc-sphingosine(200 nmol) is sonicated for 5 min with 100 /xl of 3.75 (w/v) % octylglucoside-12.5 mM dioleoylphosphatidylglycerol, made up in 1 mM DTPa, followed by the addition of 350/xl of the reaction mixture containing 120 mM HEPES buffer, pH 7.0-100 mM LiC1-25 mM MgCI2-2 mM E G T A - 2 mM dithiothreitol, and 25/zl of DG-kinase (7 mg/
[43]
RAD1OLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
517
ml). After 10 min, the reaction is started by adding 50 txl of 10 mM [32p]ATP (specific activity 1.6 × 105 cpm/nmol) in 20 mM imidazole buffer, pH 6.6-1 mM DTPA. After vigorous mixing, the reaction mixture is left at room temperature for 1 hr. Three milliliters of solvent S is added, and the reaction mixture is mixed vigorously for 1 min. The Bligh and Dyer extraction is continued by adding 350/xl of 1% perchloric acid followed by the addition of 1 ml of chloroform and 1 ml of 1% perchloric acid. Phases are separated by centrifugation (3000 rpm, 5 min) and the lower phase is transferred to a new vial. Analytical TLC-1 in solvent P shows formation of [32p]N-Bocsphingosine-l-phosphate (Rf = 0.42) in -40% yield. The organic phase is dried under nitrogen, 0.5 ml of 50% trifluoroacetic acid in methylene chloride is added with vigorous stirring, and reaction mixture is left at room temperature for 15 min. Solvents are evaporated under nitrogen and 3 ml of solvent S is added. The reaction mixture is sonicated for 15 rain, and chloroform (2 ml) and 1 N KC1 (2 ml) are added. The pH of the reaction mixture is adjusted to 11 by adding 50 /xl of concentrated ammonium hydroxide. Phases are separated by centrifugation (3000 rpm, 5 min) and analyzed by TLC-1 in solvent P. The lower phase (-90% of the total radioactivity) contains almost pure [32p] sphingosine-l-phosphate (Rf = 0.34). The upper phase contains [32P]sphingosine-l-phosphate (Rf -- 0.34) and unhydrolyzed [32P]N-Boc-sphingosine-l-phosphate (Rf = 0.46) in 1 : 1 proportion. The upper phase is acidified to pH 1 and extraction is repeated. After this step, the upper acidic phase contains [32p]N-Boc-sphingosine-1phosphate and the lower acidic phase [32p]sphingosine-1-phosphate. Lower phases from both extractions are combined and evaporated in vacuo to provide [32p]sphingosine-l-phosphate in -30% of the theoretical yield. The product is characterized by TLC-1 in solvents P and M and is 93% pure. Following this procedure, but using L-threo-N-Boc-sphingosine as a substrate, the corresponding L-threo-[32p]sphingosine-l-phosphate can be prepared in 35% yield.
Preparation of [4,5-3H]Sphinganine-l-phosphate This section presents the enzymatic method for preparation of [4,53H]sphinganine-l-phosphate by phospholipase D action by the method described by Van Veldhoven and Mannaerts. 14 [3H]lysodihydro-SM (40/xmol) is treated with 750 U phospholipase D from Streptornyces chromofuscus in 8 ml of 50 mM ammonium acetate buffer, pH 8.0, at 30°. After 1 hr, the insoluble reaction product is removed by centrifugation. The supernatant is allowed to react for another hour, cooled to 4 °, and centrifuged. The pellets are combined, dispersed in water (4 ml) by sonication, and cooled to 4°, and this precipitate is sedimented by centrifugation. These steps are repeated, and the final pellet is dried under
518
[44]
CHEMICAL AND ENZYMATIC SYNTHESES
vacuum and then dispersed in acetone by sonication. After cooling to 4 °, the solution is centrifuged and the supernatant is removed. This step is repeated, and the acetone-insoluble material is dried under nitrogen, dissolved in chloroform-methanol (1 : 1, v/v) by sonication, cooled to 4 °, centrifuged, and the supernatant removed. This step is repeated and the final product is dissolved in methanol (approximately 0.15 mM) and is characterized by TLC-1 in solvents L (RI = 0.26) and M ( R f = 0.34) to be -80% pure. Acknowledgment Supported in part by N I H Grant GM-43825.
[44] S y n t h e s i s o f K e y P r e c u r s o r s of Radiolabeled Sphingolipids
By
ALICJA BIELAWSKA, ZDZISLAW SZULC, and Yusvv
A. HANNUN
Introduction* This article presents synthetic methods used in the preparation of intermediates for sphingolipids (SLs) discussed elsewhere in this volume. 1,2 We * Sphingolipid nomenclature: Sphingosine: 4E-octadecene-l,3-diol-2-amino [CA: 123-78-4] contains two chiral centers on carbon atoms C-2 and C-3 that create four stereoisomers: an enantiomeric pair of the erythro and threo diastereoisomers. Extending geometrical (4E) "trans" isomer to its "cis" (4Z) isomer, sphingosine can create eight stereoisomers. There is a great deal of confusion regarding the DL nomenclature of optical isomers of sphingolipids, particularly the threo isomers, as both chiral centers have been chosen as a point of reference of sphingolipid chirality. 2~,2b Using DL nomenclature, the natural erythro-sphingosine is (2D,3D) and its enantiomer is (2L,3L). The threo isomers are (2D,3L) and (2L,3D). Using chiral carbon C-2 as a reference, the (2D,3L) isomer is D-threo, but using chiral carbon C-3 as a reference this isomer is L-threo. To avoid these confusions, we have decided to n a m e the absolute configuration of sphingolipids using the (CIP) system for stereochemical assignment of the chiral c o m p o u n d s 2c and keep the 3-D and 3-L nomenclature. 2~ Following these rules, D-erythro-sphingosine (2D,3D) constitutes (2S,3R) configuration. The remaining sphingosine stereoisomers are L-erythro-, (2L,3L), (2R,3S); L-threo-, (2D,3L), (2S,3S); and D-threo-, (2L,3D), (2R,3R). i A. Bielawska, Y. A. H a n n u n , and Z. Szulc, Methods Enzymol. 311 [42] 1999 (this volume). 2 A. Bielawska and Y. A. H a n n u n , Methods" Enzyrnol. 311 [43] 1999 (this volume). 2a H. E. Carter, D. S. Galanos, and J. Fujino, Can. J. Biochem. Physiol. 34, 320 (1956). 2b W. Stoffel and G. Sticht, Hoppe-Seyler's Z. Physiol. Chem. 348, 1561 (1967). 2c E. L. ElM, S. H. Wilen, and L. N. Mander, "Stereochemistry of Organic C o m p o u n d s . " Wiley, New York, 1994.
METHODS IN ENZYMOLOGY,VOL. 311
Copyrighl © 1999by AcademicPress All rights of reproduction in any form reserved. 0076-6879/99$30.00
RADIOLABELED
CERAMIDES
AND PHOSPHOSPHINGOLIPIDS
499
[43] P r e p a r a t i o n o f R a d i o l a b e l e d C e r a m i d e s and Phosphosphingolipids By ALICJA B1ELAWSKAand YUSUF A. HANNUN Introduction Radiolabeled sphingolipids (radio-SLs) with high specific activity, labeled at designated parts of the molecule, allow thorough investigation of their metabolic fate and quantitation of enzyme activities in sphingolipid metabolic pathways. To prepare structurally homogeneous radio-SLs, welldesigned synthetic approaches and precursors should be employed, which are available only in the course of stereospecific synthesis. In general, a few methods can be considered for the preparation of radiolabeled phospho-SLs: (1) biosynthetic approach using radioactive precursors such as L-serine, fatty acids, choline, or 32p. This approach generally results in low activity and leads to mixtures. (2) Labeling in the course of total synthesis following established synthetic methods. (3) Synthetic or enzymatic approaches that are mainly limited to the transformation of existing functional groups present in the structure of natural and synthetic phospho-SLs. This latter approach was commonly used during the early stage of sphingolipid biochemistry and, due to its simplicity and effectiveness, still remains a "method of choice" for many radiolabeled analogs. Homogeneous simple synthetic radio-SLs are available at the level of sphingosine and ceramide stereoisomers. Unfortunately, to date, full access to structurally homogenic complex radio-SLs is limited because of deficiency of the suitable synthetic precursors. Radioactive complex SLs are mainly reconstructed from their natural forms. We would like to stress two general problems encountered with the utilization of natural SLs as precursors in the radiolabeled synthesis. First, naturally occurring SLs exist as nonhomogeneous mixtures due to variation in the length, saturation, and hydroxylation of the sphingoid backbone and the N-acyl groups. Second, the commonly applied procedures for the isolation and preparation of simple SLs from complex precursors (such as lysosphingomyelin from sphingomyelin) can lead to a mixture of erythro- and threo-diastereoisomers due to epimerization at the C-3 position, a result of the tendency of the allylic alcohol to racemize during acidic hydrolysis. This article presents synthetic and enzymatic methods for the preparai K. A. Karlsson,
Chem. Phys. Lipids
METHODS IN ENZYMOLOGY, VOL, 311
5, 6 ( 1 9 7 0 ) .
Copyright © 1999 by Academic Press All rights of reproduction in any lorm reserved. 0076-6879/99 $30.00
500
CHEMICAL AND ENZYMATIC SYNTHESES
[43]
tion of ceramide, SM, and ceramide phosphate labeled in the N-acyl group; SM and lyso-SM labeled in the choline group; ceramide phosphate and sphingosine phosphate labeled in the phosphate group; and the combination thereof leading to multiple-labeled analogs. Materials [3H]Sodium borohydride (450 mCi/mmol), [3H]acetic anhydride (85 mCi/mmol), [1-14C]hexanoic acid (55 mCi/mmol), N-hydroxysuccinimidyl ester of [1-14C]hexanoic acid (50 mCi/mmol), [9,10-3H]palmitic acid (50 Ci/ mmol), [1J4C]palmitic acid (54.2 Ci/mmol), [14C]methyl iodide (55.7 mCi/ mmol), and [3H]methyl iodide (82 Ci/mmol) are obtained from American Radiolabeled Chemicals (St. Louis, MO). Trifluoroacetic acid (TFA) and phytosphingosine hydrochloride are from Sigma. Amberlite IRA 400 (OH-) resin and palmitic acid are from Fluka Chem. Corp. (Milwaukee, WI). CH3COONa × 3H20, cyclohexylamine, diisopropylethylamine, Na2S203, and diethylphosphoryl cyanide (DEPC) are from Aldrich Chemical Company, Inc. (Milwaukee, WI). TLC plates: The following TLC plates are used as indicated in the specific sections: (1) TLC-1; precoated TLC sheets of silica gel 60 F 254 (0.2 layer thickness) are from EM Separation Technology, (2) TLC-2; precoated thin-layer plates Kieselgel 60 (0.25 mm layer thickness) are from EM Separation Technology, (3) TLC-3; precoated thin-layer plates PK5 silica gel 150 A (0.5 mm layer thickness) are from Whatman Laboratory Division, and (4) TLC-4; Linear-K preadsorbent strip, silica gel 150A (1.0 mm layer thickness) are from Whatman Laboratory Division. Silica gel 60 (particle size 0.040-0.063 mm) (230-400 Mesh ASTM) for column chromatography CAS 63231-67-4 is from EM Separation Technology. Sep-Pac Plus Silica, Sep-Pac Plus CM, and Sep-Pac Plus C18 cartridges are from Millipore Corporation, Waters Chromatography Division (Milford, MA). All solvents are of analytical grade or better and are obtained from Mallinckrodt, Burdick and Jackson, or J. T. Baker. Anhydrous N, N-dimethylformamide: DMF (packaged in prescored ampoules) is from Aldrich Chemical Company, Inc. General Methods Used during Synthesis Tetrahydrofuran (THF) is freed of peroxides by passing over basic alumina, followed by saturation with dry argon. Cyclohexylamine, diisopropylethylamiue, and diethylphosphoryl cyanide are distilled before using.
[43]
RADIOLABELED CERAM|DES AND PHOSPHOSPHINGOLIPIDS
501
Key Precursors for Preparation of Radiolabeled Sphingolipids and Analytical Standards Stereoisomers of sphingosine, sphinganine, and their N-tert-butoxycarbonyl and N-acyl derivatives, N-hydroxysuccinimidyl ester of [1-UC]hexa noic acid, ceramide-l-phosphoryl-N,N-dimethylethanolamine, and sphingosylphosphorylamine are synthesized according to the procedures presented elsewhere in this volume. 2 Preparation of stereoisomers of [3-3H]sphingosine and [3-3H]ceramide is presented elsewhere in this volume. 3
Thin-Layer Chromatography Purity of the compounds, the reaction progress, and chromatographic elution profiles are checked routinely by thin-layer chromatography using analytical TLC plate: TLC-1. Purification is performed using preparative TLC plates: TLC-2, -3, or -4.
Flash Column Chromatography Separation of reaction products and purification of the obtained compounds are achieved by flash chromatography under positive pressure of nitrogen gas as described previously. 4 Fractions containing impure material are rechromatographed.
Solvent Systems The solvent systems used for TLC and/or flash column chromatography purification are (A) chloroform-methanol-2 N ammonium hydroxide (4 : 1 : 0.1, v/v); (B) methylene chloride-methanol (93 : 7, v/v); (C) chloroform-methanol-15 mM CaC12 (60:35:8, v/v); (D) chloroform-methanol (19 : 1, v/v); (E) chloroform-methanol-water (65 : 25 : 4, v/v); (F) chlorof o r m - m e t h a n o l - a m m o n i u m hydroxide-water (160 : 40 : 1 : 3, v/v); (G) chloroform-methanol-concentrated ammonium hydroxide (65 : 35 : 8, v/v); (H) chloroform-methanol-ammonium hydroxide-water (65 : 35 : 2.5 : 2.5, v/v); (I) chloroform-methanol-water (3 : 48 : 47, v/v); (J) chloroform-methanolwater (86 : 14 : 1, v/v); (K) methanol (containing 7% water and 1% concentrated ammonium hydroxide, v/v) and chloroform (1 : 1, v/v); (L) chloroform-methanol-water (60:35:8, v/v); (M) n-butanol-acetic acid-water (3 : 1 : 1, v/v); (N) chloroform-methanol-ammonium hydroxide (60 : 30 : 5, 2 A. Bielawska,Z. Szulc, and Y. A. Hannun, MethodsEnzymol. 311 [44] 1999 (this volume). 3 A. Bielawska,Y. A. Hannun, and Z. Szulc,Methods Enzymol. 311 [42] 1999 (this volume). 4W. C. Still, M. Kahn, and A. Mitra, J. Org. Chem. 43, 2923 (1978).
502
CHEMICALAND ENZYMATICSYNTHESES
[43]
v/v); (P) c h l o r o f o r m - a c e t o n e - m e t h a n o l - a c e t i c acid-water (10 : 4 : 3 : 2 : 1, v/v); (R) chloroform-methanol (2:1, v/v); and (S) chloroform-methanol (1:2, v/v). Analytical Assays C o m p o s i t i o n and Diastereoisomeric Purity o f Sphingoid Bases
The purity of sphingosine, sphinganine, and their radiolabeled analogs is evaluated by high-performance liquid chromatography (HPLC) using eicosasphingosine (C20-sphingosine) as an internal standard, as described by Merrill et al. 5 Radioactivity M e a s u r e m e n t
Radioactivity is measured on a Wallac LKB 1214 Rackbeta liquid scintillation counter with High Flash Point Cocktail Safety-Solve (Res. Product Int. Corp. Mount Prospect, IL) as the scintillation fluid. Radioactivity Detection
Detection and quantitation of the radioactivity of the obtained radiolabeled compounds are performed by exposing TLC plates containing radioactive compounds to TLC/Scanner (System 200 Imaging Scanner, Bioscan, Inc. Washington, DC) or to Phosphoimager (Molecular Dynamics, Sunnyvale, CA) Analytical TLC plates containing tritium can be exposed to radiography [after using surface autoradiography enhancer (En3Hance spray, NEN Research Products, Boston, MA]. L i p i d Extractions
Lipid extractions from the crude reaction mixtures are performed using the Bligh and Dyer extraction method 6 or the Folch et al. 7 procedure. Phosphorous measurement is performed using a published method. ~ DG-kinase phosphorylation is performed using a published methodg; this method is also presented in volume 312. l° A. H. Merrill, Jr., E. Wang, R. E. Mullins, W. C. L. Jamison, S. Ninkar, and D. Liotta, Anal Biochem. 171, 373 (1998). 6E. A. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 7j. Folch, M. Lees, and G. H. Sloane-Stanley,J. Biol. Chem. 226, 497 (1957). 8B. N. Ames, Methods' EnzymoL 8, 115 (1966). 9p. p. Van Veldhoven, W. R. Bishop, D. A. Yurivich, and R. M. Bell, Biochem. Mol. Biol. Inter. 36, 21 (1995). 10D. Perry, A. Bielawska, and Y. A. Hannun, Methods' Enzymol. 312 (1999).
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIP1DS
503
OH X - C(O)([3HICH2)nCH3
R2OA~'~'~N (CH2)12C H3 NH-C(O)([3H]CH2)nCH3
OH R2OA~"~
(CH2)12CH3 - -
NI-I2
OH X -14C(O)(CH2)nCH3
R2 = H, phosphate, choline phosphate or oligosaccharide chain X = H, C1, active ester group
~ R20
(CH2)12CH3 NH- C(O)(CH2)nCH3
Fl(~. 1. Approach to the synthesis of [3H]- and [14C]sphingolipidslabeled in the N-acyl group.
P r e p a r a t i o n of S p h i n g o l i p i d s L a b e l e d i n t h e A m i d e M o i e t y
Principles C e r a m i d e a n d its m o r e c o m p l e x derivatives are synthesized f r o m the c o r r e s p o n d i n g s p h i n g o i d bases p r e p a r e d synthetically,2 n a t u r a l l y , 1~-16 or from lyso-SLs by classical m e t h o d s used in p e p t i d e chemistry. S t r i n g e n t c o m p e t i t i o n in the reactivity of the a m i n o a n d hydroxyl groups in SLs t o w a r d O- a n d N - a c y l a t i o n favors N - a c y | a t i o n products. T h e a m i d e b o n d is usually p r e p a r e d by the action of acid a n h y d r i d e s 17,18 or acid halides 192° by t r a n s a c y l a t i o n of the activated fatty acids as N - h y d r o x y s u c c i n i m i d e esters, 2L22 or by p - n i t r o p h e n y l e s t e r s Y A l t e r n a t i v e m e t h o d s have b e e n H R. C. Gaver and C. C. Sweeley, J. Am. Oil Chem. Soc. 42, 294 (1965), 12R. Cohen, Y. Barenholtz, S. Gan, and A. Dagan, Chem. Phys. Lipids 35, 371 (1984). 13p. p. Van Veldhoven, R. J. Foglesong, and R. M. Bell, J. Lipid Res. 30, 611 (1989). ~4p. p. Van Veldhoven and G. P. Mannaerts, J. Biol. Chem. 266, 12502 (1991). ~5M. Buneman, K. Liliom, B. T. Brandts, L. Pott, J. L. Tseng, D. M. Desiderio, G. Sun, D. Miller, and G. Tigyi, E M B O J. 15, 101 (1996). ~ A. Kisic, M. Tsuda, R. J. Kulmacz, W. K, Wilson, and G. J. Schroepfer, J. Lipid Res. 36, 787 (1995). 17R. C. Gaver and C. C. Sweeley, J. Am. Chem. Soc. 88, 3643 (1966). ~ A. Bielawska, H. M. Crane, D. Liotta. L. M. Obeid, and Y. A. Hannun, J. BioL Chem. 268, 26226 (1993). 19K. C. Kopaczyk and N. S. Radin, J. Lipid Res. 6, 140 (1965). 2, A. Bielawska, D. Perry, S. Jayadev, C. McKay, J. Shayman, and Y. A. Hannun, J. Biol. Chem. 271, 12642 (1996). 21 y. Lapidot, S. Rapport, and Y. Wolman, J. Lipid Res. 8, 142 (1967). 27A. Futerman and R, E. Pagano, Methods Enzymol. 209, 437 (1986). 2:tB. Neises, T. Andries, and W. Steglich, J. (?hem. Soc. Chem. Commun. 1132, (1982).
504
CHEMICALAND ENZYMATICSYNTHESES
[43]
developed using coupling agents such as N-ethoxycarbonyl-2-ethoxy-l,2dihydroquinoline, 24 dicyclohexylcarbodiimide (DCC), 12 diphenyl phosp h o r a z i d a t e y 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,26'27 ethylchIoroformate, 28 pentafluorophenol, 29 or diethylphosphoryl cyanide ( D E P C ) ? ° A new enzymatic method for the preparation of radioactive ceramides using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase (SCDase) has been published? l Four approaches for the radio acylation of the amino group of sphingoid bases employ (1) acetic anhydride, (2) acyl chlorides, (3) N-hydroxy succinimidyl esters, and (4) fatty acids and D E P C as a coupling agent. To synthesize C2-ceramides, use the first approach; to prepare long chain ceramides, use the second approach; and to synthesize C6- and Cs-ceramides, use the last approach. The D E P C method employs diethylphosphoryl cyanide as a coupling agent in the presence of triethylamine and is rapid, free of racemization, utilizes acids without prior derivatization, and generates relatively pure products. We have noticed that this reaction needs to be carried out in anhydrous conditions, under nitrogen atmosphere, and at 0 °. The use of freshly distilled D E P C and triethylamine is critical. Following these rules we have synthesized all stereoisomers of [14C]C6- and [14C]dihydro-C6ceramides with about 90% yield. The acid chloride method (the second approach), which we have named the "one pot" procedure, employs a simple and effective protocol of in situ generation of the acid chlorides from fatty acids and thionyl chloride or oxalyl chloride ~9'2° following the introduction of a THF-aqueous CH3COONa reaction system and selected sphingosines. Using this method, stereoisomers of various radiolabeled long chain ceramide and dihydroceramides can be synthesized with 70-85% yield.
24Z. Dong and J. A. Butcher, Jr., Chem. Phys. Lipids 66, 41 (1993). 25S. Yamada, N. Ikota, T. Shioriand, and S. Tachibana, J. Am. Chem. Soc. 97, 7174 (1975). 26S. Hammarstrom, J. Lipid Res. 12, 760 (1971). 27T. Levade, S. Gatt, A. Maret, and R. Salvayre,J. Biol. Chem. 266, 13519 (1991). 2s D. Aquotti, S. Sonnino, M. Masserine, L. Casella, G. Fronza, and G, Tenamanti, Chem. Phys. Lipids 40, 71 (1986). 29R. Jennemann, C. Gnewuch, S. Bolets, B, L. Bauer, and H. Wiegandt, J. Biochem. 115, 1947 (1994). 3oj. K. Anand, K. K. Sadozai, and S.-I. Hakamori, Lipids 30, 995 (1996). 31S. Mitsutake, K. Kita, N. Okino, and M. Ito, Anal Biochem. 247, 52 (1997).
[43]
RAD1OLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
505
Preparation of Radiolabeled Ceramides
Synthesis of N-[1J H-acetyl]-D-erythro-sphingosine: N-J1J H]D-erythro-CTceramide This section presents the synthesis of stereoisomers of C2-ceramide and C2-dihydroceramide and the preparation of N-acetylphytosphingosine, all labeled in the acetyl group, by a method 18 adapted from the procedure for the preparation of nonradioactive C2-ceramide. 17'j8 [3H]Acetic anhydride (5 mCi, 59 /xmol) is cooled in an ice bath and diluted with 100/xmol (5/xl) of cold acetic anhydride in 5 ml of dry methanol. D-erythro-Sphingosine (12 rag, 40 txmol) is dissolved in 2 ml of dry methanol and is added to the methanol solution of [3H]acetic anhydride. The reaction mixture is stirred in an ice bath for 4 hr, when the TLC-1 solvent shows that the reaction is complete. Cold water (5 ml) is added dropwise to produce a white precipitate, and the mixture is stirred in an ice bath for an additional 30 rain. The precipitate is separated by centrifugation (3000 rpm, 5 min, 5°), separated from the supernatant, dissolved in 5 ml of chloroform, and washed with 1 ml of 0.1 N NaOH. Phases are separated by centrifugation, and the organic phase is washed with water until no radioactivity is present in the upper phase. The organic phase is dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-4 in solvent A. The area corresponding to C2-ceramide is scraped, and ceramide is extracted three times with 5 ml of solvent R. The extraction efficiency is evaluated quantitatively by scintillation counting. Combined extracts are dried under vacuum to give 12.5 mg (91.5% yield) of [3H]I>erythro-C2ceramide (specific activity: 15.75 mCi/mmol). The purity of this product (-96% pure) is established by analytical TLC-1 in solvent B (Rf = 0.21) and in solvent A (Rf = 0.64). This product is dissolved in solvent R at 10 mM and is stored at - 2 0 ° for several months. Using the just-described procedures, the remaining C2-ceramide and C2-dihydroceramide stereoisomers--L-threo-(2S,3S),D-threo-(2R,3R), and L-erythro-(2R,3S)--can be prepared starting from the corresponding sphingosine and sphinganine isomers. [3H]C2-ceramides/dihydroceramides labeled in the sphingosine/sphinganine backbone can be prepared by the acylation of [3-3H]sphingosine and [3-3H] - or [4,5-3H]sphinganines with unlabeled acetic anhydride following the just-described procedure.
Synthesis of N-[1-3H-acetyl]phytosphingosine: N-[1--~H] C2-phytocerarnide [3H]Acetic anhydride (5 mCi, 59 p~mol) is cooled down in an ice bath and diluted with 100/xmol (5 ~1) of cold acetic anhydride in 5 ml of dry methanol. Phytosphinogosine hydrochloride (14.2 mg, 40/~mol) is dissolved
506
CHEMICAL AND ENZYMATIC SYNTHESES
[43]
in 2 ml of dry methanol, and the pH is adjusted to 9 with 2 N NaOH in methanol and added to the methanol solution of [3H]acetic anhydride. The reaction mixture is stirred in an ice bath for 4 hr. TLC-1 in solvent A shows that the reaction is complete. The product is isolated and purified following the procedure developed for the C2-ceramide preparation. After final purification, [3H]C2-phytoceramide is obtained in 77% yield (12.2 mg, specific activity: 15.75 mCi/mmol), with a purity of -98% as established by analytical TLC-1 in solvent A ( R f = 0.4).
Synthesis of N-[l YC-Hexanoyl]-D-erythro-sphingosine: N-[1J4C]-D-erythro-C6-ceramide This section presents the synthesis of stereoisomers of C6-ceramide and C6-dihydroceramide by methods that have been adapted from general procedures for the preparation of radioactive ceramides by the DEPC method 3° and by the NHS ester method. 21'22 Via the DEPC Method. I>erythro-Sphingosine (4.9 mg, 16.4/xmol), in freshly distilled and dry DMF-methylene chloride 1:3 mixture (2 ml), is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing [1J4C]hexanoic acid (0.9 mCi, 16.4 txmol) in 900 /xl of dry methylene chloride followed by the addition of 32.8/xmol (11 txl) of freshly distilled DEPC. The volume of the solvents is adjusted to 11 ml, keeping the proportion DMF-methylene chloride as 1 : 3 (v/v). After flushing with argon, the reaction mixture is cooled to 0°, and freshly distilled triethylamine (16.4/xmol, 9/xl) is added under argon. Stirring under argon is continued for 2 h, solvents are evaporated, and the product is extracted by the Bligh and Dyer 6 procedure. The upper phase is reextracted with 2 ml of chloroform; liquid scintillation counting detects almost no radioactivity in the upper phase. TLC analysis of the radioactive organic phase is performed in solvent B (C6-ceramide: Rf = 0.34) and in solvent A (C6-ceramide, R f = 0.74). Solvent A, which separates well ceramides from unreacted fatty acids and sphingosine, shows that -80% of radioactivity is incorporated into ceramide. The organic phase is dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-4 in solvent A. The area corresponding to C6-ceramide is scraped, and ceramide is extracted three times with solvent R. Extraction efficiency is evaluated by liquid scintillation counting. Combined extracts are dried under vacuum to give 5.54 mg (85% theoretical yield) of N-[1-~4C]D-erythro-C6-ceramide. The purity of this product was -95% as established by analytical TLC-1 in solvents C and A. This product is dissolved in solvent R at 10 mM (specific activity: 1.22 × 105 cpm/nmol) and stored at - 2 0 ° for several months.
[431
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
507
Using the just-described procedures, the remaining C6-ceramide and C6-dihydroceramide stereoisomers--L-threo-( 2S,3S), D-threo-( 2R,3 R ), and berythro-(2R,3S) can be prepared starting from the corresponding sphingosine and sphinganine isomers. Via the NHS Ester Method. D-erythro-Sphingosine (6.0 mg, 20/xmol) in 1 ml of dry DMF is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing N-hydroxysuccinimidyl ester of [1-14C]hexa noic acid (1.0 mCi, 20/xmol) in 1 ml of dry DMF followed by the addition of 10/xl of freshly distilled diisopropylethylamine. The reaction is allowed to proceed at room temperature under nitrogen atmosphere in the dark, with stirring. Progress is monitored periodically by TLC-1 in solvent C. After 15 hr, the reaction is almost complete, the reaction mixture is acidified with 20/xl of 3 N HC1, and the mixture is dried completely under a stream of nitrogen. The product is extracted by the Bligh and Dyer 6 procedure. The upper phase is reextracted with 2 ml of chloroform, dried under nitrogen gas, dissolved in solvent R, and purified by preparative TLC-3 in solvent E or F. The area corresponding to C6-ceramide is scraped, and ceramide is extracted three times with 5 ml of solvent R. The extraction efficiency is evaluated by liquid scintillation counting. The combined extracts are dried under vacuum to give 6.4 mg (80% theoretical yield) of N-[1-14C]D-erythroC6-ceramide. The purity of this product is -95% as was established by analytical TLC-1 in solvents C and A.
Synthesis of N-[3H or NC-palrnitoyl]D-erythro-sphingosine: N-[9,10-~H]D erythro-C i6-ceramide or N-[1-14C] D-erythro-C m-ceramide This section presents the synthesis of stereoisomers of C16-ceramide and C16-dihydroceramide via an acyl chloride method that has been adapted from the procedures for the preparation of nonradioactive ceramides and their analogs. ~9'2° [9,10-3H]Palmitic acid (4.8 mCi, 0.08/xmol), diluted with 8/xmol of cold palmitic acid, is refluxed with 0.5 ml of oxalyl chloride in dry benzene for 1 hr under nitrogen atmosphere. 16The solution is then evaporated to dryness under vaccum. D-erythro-Sphingosine (2.6 mg, 8 txmol) dissolved in 2.5 ml of THF is added via syringe to the crude [3H]palmitic chloride followed by the addition of 1.25 ml of 50% CH3COONa. The reaction mixture is stirred vigorously for 4 h. Ceramide is isolated by partitioning with chloroform (6 ml)-methanol (3 ml)-water (2.25 ml). The phases are separated, and the lower phase (8 ml) is washed twice with 2 ml of water. More than 90% of the theoretical radioactivity (calculation from the specific activity of the original [3H]palmitic acid) is recovered in the lower organic phase with almost no radioactivity in the upper phase. TLC-1 analysis of the
508
CHEMICAL AND ENZYMATIC SYNTHESES
[43]
organic phase, performed in solvent B (separates ceramides from esterified ceramides) and in solvent A (separates ceramides from unreacted fatty acids and sphingosine), shows that -90% of radioactivity is incorporated into N-[9,10-3H]D-erythro-C16-ceramide. Ceramide is purified by preparative TLC-3 in solvent A, the area corresponding to C16-ceramide is scraped, and ceramide is extracted three times with solvent R. The extraction efficiency is monitored by liquid scintillation counting. The combined extracts are dried under vacuum and repurified with solvent B. The final [3H]Derythro-C16-ceramide (82% yield) is dissolved in solvent R at 10 mM (specific activity: 1.3 × 105 dpm/nmol). The purity is -96%, as established by analytical TLC-1 in solvent B (Rf = 0.21) and in solvent A (Rf = 0.64). This procedure can be used to prepare the remaining C16-ceramide and C~6-dihydroceramide isomers and other Cn-ceramides.
Preparation of Sphingomyelin Labeled in the N-Acyl Group
Synthesis of N-[1-14C-Hexanoyl]-D-erythro-sphingosylphosphorylcholine: N-[1-NC]-C6-SM Sphingomyelin labeled in the N-acyl group is obtained by N-acylation of D-erythro-sphingosylphosphorylcholine via the DEPC method or via the NHS ester method, following procedures developed for N-[1-~4C]ceramide preparation. 26,34 D-erythro-Sphingosylphosphorylcholine (6.0 mg, 13 /xmol) in 2 ml of dry DMF is added via syringe to a screw-capped, Teflon-sealed, air-evacuated vial containing N-hydroxysuccinimidyl ester of [1J4C]hexanoic acid (0.65 mCi, 13/xmol) in 1 ml of dry DMF followed by the addition of 10 /xl of freshly distilled diisopropylethylamine. The reaction is allowed to proceed at room temperature under nitrogen atmosphere in the dark, with stirring. Progress is monitored periodically by TLC-1 in solvent C. After 25 hr the reaction is almost complete, and the reaction mixture is acidified with 20/xl of 3 N HC1 and then dried completely under a stream of nitrogen. The product is extracted by the Bligh and Dyer procedure. The upper phase is reextracted with 2 ml of chloroform, dried under nitrogen, dissolved in solvent R, and purified by preparative TLC-3 in solvent E or F. The area corresponding to C6-SM is scraped, and sphingomyelin is extracted five times with 5 ml of solvent R. The extraction efficiency is evaluated by liquid scintillation counting. The combined extracts are dried under vacuum to give 5.3 mg (75% theoretical yield) of N-[1-14C]D-ervthro-C6-SM.The purity of this product is -95% as established by analytical TLC-1 in solvents C and G.
[43]
R A D I O L A B E L E D C E R A M I D E S A N D PHOSPHOSPH1NGOLIP1DS
509
Preparation of Radiolabeled Sphingomyelin Principles Sphingomyelin can be radiolabeled (Fig. 2) in various parts of the molecule, depending on the experimental needs. Reduction of 3-keto-SM labels the C-3 position of the sphingosine backbone ([3-3H]-SM). N-acylation of sphingosylphosphorylcholine (lyso-SM) with radioactive fatty acids generates SM labeled in the N-acyl group (N-[1J4C-acyl]-labeled SM, see procedure described earlier). The quaternization procedure of ceramidel-phosphoryl-N,N-dimethylethanolamine (demethylated-SM) generates [14C- choline]-labeled SM, and this method is presented next. Chemically homogeneous SMs are available presently in a limited scope as their syntheses were described only for a few selected D-erythro series. 32"33 These multistep methods have not been adapted and optimized yet to the needs of SM radiochemistry. A synthetic approach used by Bitmann's group 33may be considered as a method for the preparation of stereoisomers of radiolabeled SM if radiolabeled ceramide stereoisomers are used as substrates. Also, by employing the [3H]trimethylamine at the last synthetic step, [3H-choline]-SM or double-labeled-SM (in the choline and in the amide part) may be prepared. Radiolabeling in the Choline Moiety. This method was developed for natural SM 34,35 and involves the demethylation of SM to ceramide 1-phosphoryl-N, N-dimethylethanolamine (de-SM) and subsequent quaternization with radioactive [t4C]- or [3H]methyl iodide. Quaternization of de-SM with [3H]methyl iodide allows the preparation of [3H-choline]SM with a high specific activity (~80 Ci/mmol). The demethylation process is presented elsewhere in this volume. 2 Preparation o f [14C-choline]Sphingomyelin
This section presents the synthesis of [14C-choline]SM by a method that has been adapted from the procedure published by Stoffel et aL 34 and the synthesis of [14C-choline]lyso-SM, obtained from [14C-choline]SM by a method that has been adapted from previously described procedures. ~2 15 [~4C]Methyl iodide (5 mCi, 89.8/~mol) is dissolved in 2 ml of dry methanol and transferred to an air-evacuated flask containing 5 ml of milky 3~R. R. Schmidt,in "Synthesisin LipidChemistry"(H. P. Tyman,ed.), p. 93. BrunelUniversity, Uxbridge, UK, 1996. ~3H. S. Byun, R. K. Erukulla, and R. Bittman, J. Org. Chem. 59, 6495 (1994). 34W. Stoffel,D. LeKim,and T. S. Tsung, Hoppe-Seyler's Z. Physiol. Chem. 352, 1058 (1971). 3~W. Stoffel,Methods" Enzymol. 35, 533 (1975).
510
[43]
CHEMICAL AND ENZYMATIC SYNTHESES A
O ®
O II
,
®o
NHCOR 3-keto-SM
I NaB3H4 o ® II (H3C)3N~'/X,O" ~ ' O C)
s
OH
O ~ ][ JH NHCOR
(CH2)12CH 3
[3-3H]-SM
®
o
OH
II
-
(H3C)3N'v'A~"i ' ~ O ~ {=)
(CH2)12CH 3
N1t2 lyso-SM
R14COOH ®
o II
OH --
(H3C)3NV/X.O" f " O ~ (CH2)12CH3 NH14CO" " R N-[14C-acyl]-SM O II
OH -
(H3C)2NV / X - O ~ X O ~ ( C H 2 ) 1 2 C H OH
3
NHCOR
N-demethylated-SM
14CH3J o ~ Oil ---~4 e pII (H3 C ) 3 N x / / " - . X g - [ X O ~ ( C H 2 ) , 2
6
{~)
Ii
CH3
NHCOR [14C-choline]-SM
FlG. 2. (a) Approaches to the preparation of sphingomyelin radiolabeled in various parts of the molecule. (b) Approach to the synthesis of [3H-] or [14C-choline]-labeled sphingomyelin and lysosphingomyelin.
[43]
511
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
B
O II
®
OH .--NHCOR
SM I C6H5SNa DMSO,80°C (~)
~
(H3C)2HNX//X"K)"
OH
~'NO~(CH2)12CH3 NHCOR
N-demethylated-SM
I
I C3H31
14CH3I CH3
O
OH
14 ¢)1 II T-CH3- - ~ x,,/X-,O1 ~ ' O ~ CH3
8
CHo
0
(CH2)12CH3 C Hy----~~ , -
NHCOR
~'--O~(CH2)12C[ 8 NHCOR
CH3
[14C-choline]-SM
[3H-eholine]-SM acid hydrolysis
acid hydrolysis Ct-13 ,~
O
OH
~
II
v
CH3
8
NH2
®1
OH
CH3 ~
H3
O
OFl
~
II
.-=
CH3
8
NH2
®1
[3H-eholine]-Iyso-SM
[14C-choline]-Iyso-SM FIG. 2. (continued)
solution of demethylated sphingomyelin (77.3 rag, 108/.tmol) in dry methanol. This process is repeated four times followed by the addition of freshly distilled cyclohexylamine (11.1 mg, 13 ~1, 112 /,mol) in 200 /,d of dry methanol. This mixture contains more than 80% of the theoretical radioactivity of [14C]methyl iodide. The milky solution becomes progressively clear after - 1 hr and is stirred at room temperature in the dark for 24 hr. TLC1 analysis of the reaction mixture in solvent G shows formation of [14C]SM
t3
512
C H E M I C A L A N D E N Z Y M A T I C SYNTHESES
[43]
(-90% of the total radioactivity), some unidentified minor radioactive byproducts, and the ureacted substrate. Methanol is removed under a stream of nitrogen, and the reaction mixture is treated twice with 3 ml of chloroform (and redried) and dissolved in 10 ml of chloroform. The chloroform solution is washed twice with 5 ml of freshly prepared 5% Na2S203 followed by 2 N HC1 (2 × 4 ml) and water (2 × 4 ml). After centrifugation (3000 rpm, 15 rain, 5°), the upper phase is discarded and the lower phase, with the milky interphase, is diluted with chloroform to get a clear solution (15 ml total) (during the washing process, -15% of radioactivity is lost). The crude product is purified by gradient flash chromatography (11-ram-diameter column) using chloroform-methanol (65:35-5:95, v/v) as an eluting system. Each of the 5-ml fractions is monitored by counting, and elution progress is followed by TLC-1 in solvent G. Unreacted de-SM is eluted with chloroform-methanol (3 : 7, v/v); pure N-[14C-choline]SM elutes with chloroform-methanol (5 : 95, v/v). Fractions containing pure sphingomyelin are combined and evaporated under nitrogen gas to give a white solid product (45.9 rag, 3.5 mCi, 88% yield) (calculated from the specific activity of [14C]methyl iodide). Fractions containing pure de-SM are also combined and evaporated under nitrogen to give a white solid product (22 rag). [HC]Sphingomyelin is dissolved in a mixture of methanol-toluene (1:1, v/v) to yield an activity of 1 × 106 cpm/~l (8.2 mM as determined by phosphorus assay). 12 [~4C]SM can be kept at - 2 0 ° for several months without decomposition.
Preparation of F4C-choline]L ysosphingomyelin N-[~C-choline]Sphingomyelin (0.56 mCi, 6.3 rag, 8.55 /~mol) is hydrolyzed under reflux in 3.5 ml of 1 N HC1 in methanol for several hours with monitoring by TLC-1 in solvent H. After 12 hr, the reaction is complete. Solvent is evaporated and the product is partitioned into the upper phase of the Folch procedure. Briefly, the reaction mixture is redissolved in 2 ml of methanol followed by the addition of 4 ml of chloroform and 1.5 ml of water. Phases are separated and the lower phase (4 ml) is reextracted twice with 3.5 ml of solvent I. The combined upper phases contain more than 90% of [14C-choline]lyso-SM, and sphingosine and N-[~4C]phosphocholine. The pH of the combined upper phases is adjusted to 12-13 with 5 N NaOH and 12 ml of solvent J is added to transfer lyso-SM and sphingosine to the lower phase. This process is repeated three times, with the extraction efficiency evaluated by liquid scintillation counting. The lower phases are combined and evaporated in vacuo. The crude product is purified by preparative TLC-3 in solvent G. The area corresponding to N-[14C-choline]lysoSM is scraped from the plate, and the product is extracted several times
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
513
with 3 ml of solvent K. 15 Extracts containing lyso-SM are combined and evaporated under nitrogen to give a white solid product (2.5 mg, 65% yield, 0.36 mCi). Purity is assessed by TLC-1 in solvents H, L, o r N . 14
Preparation of Radiolabeled Ceramide- 1-Phosphate and Sphingosine- 1-phosphate Principles Synthesis of this class of compounds is difficult to perform, particularly for sphingosine-l-phosphate. The only practical approaches described so far for the preparation of these radiolabeled compounds are enzymatic methods. Enzymatic Methods for the Preparation of [32p, 3H or 14C]Ceramide-1phosphate and [32p or aH]Sphingosine-l-phosphate An enzymatic method employing E. coli diacylglycerol kinase (DGkinase) can be applied for the preparation of radioactive ceramide-l-phosphate (Fig. 3a) and sphingosine-l-phosphate (Fig. 3b). DG-Kinase phosphorylates diacyl and monoacyl glycerols using adenosine triphosphate (ATP) as a phosphate source. 36 This enzyme can also phosphorylate the primary hydroxyl group of ceramide9'13.20,37and their analogs. 3s This method was successfully adopted for the determination and quantitation of the endogenous ceramide in cells and tissue as well as for the fast preparation of a variety of [32p]_, [3HI' or [14C]-labeled ceramide-l-phosphates in moderate yield (-70%). DG-kinase phosphorylates only ceramides posessing the 2S configuration at the C-2 position of sphingosine backbone and generates only (2S,3R) and (2S,3S) stereoisomers. 38 We find that DG-kinase also phosphorylates N-Boc-protected Derythro-and L-threo-sphingosines and sphinganines. Based on that observation and a simple protocol for N-Boc-deprotection [mild acidic, free aminolipid liberation protocol: TFA, - 0 °, Amberlite IRA 400 (OH-), MeOH], we have successfully prepared [32p]_ o r [3H]-labeled sphingosine1-phosphate and sphinganine-l-phosphate without contamination of one stereoisomer by another? 8 The acidic hydrolysis approach, applied pre~(' J. Preiss, C. R. Loomis, W. R. Bishop, R. Stein, J. E. Niedel, and R. M. Bell, J. Biol. Chem. 261, 8597 (1986). 37 K. A. Dressler and R. N. Kolesnik, J. Biol. Chem. 265, 14917 (1990). 3~ A. Bielawska. unpublished observations.
514
[43]
CHEMICAL AND ENZYMATIC SYNTHESES
A OH HO~(CH2)12CH
3 [32p]ATP, DC-kinas~
NHCO (CH2)nCH3
NHCO (CH2)nCH 3
Ceramide
[ 32p]
OH = ATP, DG-kinase H
O
~
~
(CH2)12CH3
O II
OH =
HO ] O ~ ( C H 2 ) 1 2 C H 3 OH ~I_ll4co (CH2)nCH3
NH ' CO (CH2)nCH 3
[14C]Ceramide B
Ceramide-l-phosphate
[14C]Ceramide-1-phosphate
OH
OH
HO~(CH2)12CH3 NHBoc
N-Boc-sphingosine
HO~(CH2)
[32p] ATP, DG-kinase O HO ;HO"
O
ATP, DG-kinase O
OH
32 II
OH
II
-=
12CH3
NHBoc [ 1-3H-or 3-3H] N-Boc-Sphingosine
"(CH2)12CH3
..Px ~ ...@..~'.. HO O]HO" *" "~" ~ "(CH2)I2CH 3 NHBoc
TFA, ~0oC, ] AmberliteIRA 400 (OH-), MeOH " OH
TFA, ~0oC, [ AmberliteIRA 400 (OH-), MeOH - OH
I "~ NHBoc
~
32 II .P-. A J'-.~'-. HO ]OHO" " ~ v -(CH2)12CH3 NH 2 [ 32p] Sphingosine-l-phosphate
{
O
II
HO ] O ' ~ " ~ ' ~ ( C H 2 ) 1 2 C H 3 OH
NH 2
[1-3H - or 3-3H]
Sphingosine-1-phosphate
FIG. 3. (a) Enzymatic method for the preparation of ceramide-l-phosphate III labeled in the phosphate group or in the ceramide portion. (b) Enzymatic method for the preparation of sphingosine-l-phosphate VB labeled in the phosphate group or in the sphingosine portion. Asterisk indicates tritium labeling. (c) Enzymatic method for the preparation of ceramide-1phosphate III B and sphingosine-l-phosphate VB.
[43]
RADIOLABELED CERAMIDES AND PHOSPHOSPH1NGOLIPIDS
515
G ® (CH3)3N ~
O II --P'-. O I O1~
OH = " ..~ ~X~-*~X(CH2)12CH3 NH14COR IN-14C-acyl]-SM PL-D oH
HO ] O ' ~ ' ~ " ~ ( C H i ) l i C H 3 OH ~H14COR
[14C]-Ceramide-l-phosphate
® (CH3)3N~
0
OH
II
-
0/ ~ } N O ~ (CH2)12CI-I3 0 • 3H @ NH 2 [3-3H]-Iyso-SM PL-D
I~
I
_OH
HO I o H O ~ ( C H 2 ) 1 2 C H 3 NH 2
[3-3H]-Sphingosine- 1-phosphate F~G. 3. (continued)
viously for the generation of sphingosine-l-phosphate from ceramide phosphates, afforded sphingosine-l-phosphate as a diastereoisomeric mixture. 13,37,38 Other enzymatic methods employ bacterial phospholipase D treatment of SM or lyso-SM 13,14(Fig. 3c) or sphingosine kinase 39,4° for the preparation of radioladeled ceramide- and sphingosine-l-phosphates.
39 W. Stoffel, G. Assmann, and E. Binczek, Hoppe-Seyler's Z. Physiol. Chem. 351, 635 (1970). 4o N. Mazurek, T. Megidish, S.-I Hakamori, and Y. Igarashi, Biochern. Biophys. Res. Commun. 198, 1 (1994).
516
CHEMICAL AND E N Z Y M A T I C SYNTHESES
[43]
Preparation of N-FH-palmitoyl]o-erythro-Sphingosine-l-phosphate: FH]-D-erythro-Clo-ceramide-l-phosphate This section presents an enzymatic method for the preparation of D-
erythro-C16-ceramide phosphate and D-erythro-sphingosine-l-phosphate (labeled with 32p, 14C, or 3H) by DG-kinase action. This method has been adopted from the general method for ATP-phosphorylation of diacylglycerols and ceramides by DG-kinase. 9'36 N-[gH]Palmitoyl-D-erythro-sphingosine(0.2 mCi, 200 nmol) is sonicated for 5 min with 100/zl of 3.75% (w/v) octylglucoside-12.5 mM dioleoylphosphatidylglycerol, made up in 1 mM DTPA, followed by the addition of 350 btl of the reaction mixture containing 120 mM HEPES buffer, pH 7.0-100 mM LiC1-25 mM MgC12-2 mM E G T A - 2 mM dithiothreitol, and 25/.tl of DG-kinase (7 mg/ml). After 10 min, the reaction is started by adding 50 /xl of 10 mM ATP in 20 mM imidazole buffer, pH 6.6-1 mM DTPA. After vigorous mixing, the reaction mixture is left at room temperature for 1 hr, 3 ml of solvent S is added, and the reaction mixture is mixed vigorously for 1 min. Bligh and Dyer extraction is continued by adding 350/zl of 1% perchloric acid followed by 1 ml of chloroform and 1 ml of 1% perchloric acid. Phases are separated by centrifugation (3000 rpm, 5 rain), and the lower phase is transferred to a new vial. This organic phase is dried under nitrogen and subjected to the base cleavage in 2 ml of 0.1 N NaOH in methanol for 30 min, at room temperature. The product is extracted by the Bligh and Dyer method. The lower phase is concentrated under nitrogen gas, and the product is purified by TLC-3 in solvent P and detected using a phosphoimager. The area corresponding to [3H]D-erythro-C16-ceramide1-phosphate (Rf = 0.53) is scraped and extracted five times with 3 ml of chloroform-methanol (l :1, v/v) and is evaluated quantitatively by liquid scintillation counting. The combined exstracts are dried under vacuum to give the final product with -55% yield (calculation is made from the specific activity of the original [3H]D-erythro-C16ceramide). The product is characterized by TLC-1 in solvents P and M and is found to be 95% pure. Following this procedure, other D-erythro- and L-threo-ceramide phosphates and dihydroceramide phosphates can be prepared. However, the phosphorylation efficiency for short chain ceramides is much lower (--35% for C6-ceramide and only -15% for C2-ceramide). 4°
Preparation of o-erythro-F2p]Sphingosine-l-phosphate D-erythro-N-Boc-sphingosine(200 nmol) is sonicated for 5 min with 100 /xl of 3.75 (w/v) % octylglucoside-12.5 mM dioleoylphosphatidylglycerol, made up in 1 mM DTPa, followed by the addition of 350/xl of the reaction mixture containing 120 mM HEPES buffer, pH 7.0-100 mM LiC1-25 mM MgCI2-2 mM E G T A - 2 mM dithiothreitol, and 25/zl of DG-kinase (7 mg/
[43]
RAD1OLABELED CERAMIDES AND PHOSPHOSPHINGOLIPIDS
517
ml). After 10 min, the reaction is started by adding 50 txl of 10 mM [32p]ATP (specific activity 1.6 × 105 cpm/nmol) in 20 mM imidazole buffer, pH 6.6-1 mM DTPA. After vigorous mixing, the reaction mixture is left at room temperature for 1 hr. Three milliliters of solvent S is added, and the reaction mixture is mixed vigorously for 1 min. The Bligh and Dyer extraction is continued by adding 350/xl of 1% perchloric acid followed by the addition of 1 ml of chloroform and 1 ml of 1% perchloric acid. Phases are separated by centrifugation (3000 rpm, 5 min) and the lower phase is transferred to a new vial. Analytical TLC-1 in solvent P shows formation of [32p]N-Bocsphingosine-l-phosphate (Rf = 0.42) in -40% yield. The organic phase is dried under nitrogen, 0.5 ml of 50% trifluoroacetic acid in methylene chloride is added with vigorous stirring, and reaction mixture is left at room temperature for 15 min. Solvents are evaporated under nitrogen and 3 ml of solvent S is added. The reaction mixture is sonicated for 15 rain, and chloroform (2 ml) and 1 N KC1 (2 ml) are added. The pH of the reaction mixture is adjusted to 11 by adding 50 /xl of concentrated ammonium hydroxide. Phases are separated by centrifugation (3000 rpm, 5 min) and analyzed by TLC-1 in solvent P. The lower phase (-90% of the total radioactivity) contains almost pure [32p] sphingosine-l-phosphate (Rf = 0.34). The upper phase contains [32P]sphingosine-l-phosphate (Rf -- 0.34) and unhydrolyzed [32P]N-Boc-sphingosine-l-phosphate (Rf = 0.46) in 1 : 1 proportion. The upper phase is acidified to pH 1 and extraction is repeated. After this step, the upper acidic phase contains [32p]N-Boc-sphingosine-1phosphate and the lower acidic phase [32p]sphingosine-1-phosphate. Lower phases from both extractions are combined and evaporated in vacuo to provide [32p]sphingosine-l-phosphate in -30% of the theoretical yield. The product is characterized by TLC-1 in solvents P and M and is 93% pure. Following this procedure, but using L-threo-N-Boc-sphingosine as a substrate, the corresponding L-threo-[32p]sphingosine-l-phosphate can be prepared in 35% yield.
Preparation of [4,5-3H]Sphinganine-l-phosphate This section presents the enzymatic method for preparation of [4,53H]sphinganine-l-phosphate by phospholipase D action by the method described by Van Veldhoven and Mannaerts. 14 [3H]lysodihydro-SM (40/xmol) is treated with 750 U phospholipase D from Streptornyces chromofuscus in 8 ml of 50 mM ammonium acetate buffer, pH 8.0, at 30°. After 1 hr, the insoluble reaction product is removed by centrifugation. The supernatant is allowed to react for another hour, cooled to 4 °, and centrifuged. The pellets are combined, dispersed in water (4 ml) by sonication, and cooled to 4°, and this precipitate is sedimented by centrifugation. These steps are repeated, and the final pellet is dried under
518
[44]
CHEMICAL AND ENZYMATIC SYNTHESES
vacuum and then dispersed in acetone by sonication. After cooling to 4 °, the solution is centrifuged and the supernatant is removed. This step is repeated, and the acetone-insoluble material is dried under nitrogen, dissolved in chloroform-methanol (1 : 1, v/v) by sonication, cooled to 4 °, centrifuged, and the supernatant removed. This step is repeated and the final product is dissolved in methanol (approximately 0.15 mM) and is characterized by TLC-1 in solvents L (RI = 0.26) and M ( R f = 0.34) to be -80% pure. Acknowledgment Supported in part by N I H Grant GM-43825.
[44] S y n t h e s i s o f K e y P r e c u r s o r s of Radiolabeled Sphingolipids
By
ALICJA BIELAWSKA, ZDZISLAW SZULC, and Yusvv
A. HANNUN
Introduction* This article presents synthetic methods used in the preparation of intermediates for sphingolipids (SLs) discussed elsewhere in this volume. 1,2 We * Sphingolipid nomenclature: Sphingosine: 4E-octadecene-l,3-diol-2-amino [CA: 123-78-4] contains two chiral centers on carbon atoms C-2 and C-3 that create four stereoisomers: an enantiomeric pair of the erythro and threo diastereoisomers. Extending geometrical (4E) "trans" isomer to its "cis" (4Z) isomer, sphingosine can create eight stereoisomers. There is a great deal of confusion regarding the DL nomenclature of optical isomers of sphingolipids, particularly the threo isomers, as both chiral centers have been chosen as a point of reference of sphingolipid chirality. 2~,2b Using DL nomenclature, the natural erythro-sphingosine is (2D,3D) and its enantiomer is (2L,3L). The threo isomers are (2D,3L) and (2L,3D). Using chiral carbon C-2 as a reference, the (2D,3L) isomer is D-threo, but using chiral carbon C-3 as a reference this isomer is L-threo. To avoid these confusions, we have decided to n a m e the absolute configuration of sphingolipids using the (CIP) system for stereochemical assignment of the chiral c o m p o u n d s 2c and keep the 3-D and 3-L nomenclature. 2~ Following these rules, D-erythro-sphingosine (2D,3D) constitutes (2S,3R) configuration. The remaining sphingosine stereoisomers are L-erythro-, (2L,3L), (2R,3S); L-threo-, (2D,3L), (2S,3S); and D-threo-, (2L,3D), (2R,3R). i A. Bielawska, Y. A. H a n n u n , and Z. Szulc, Methods Enzymol. 311 [42] 1999 (this volume). 2 A. Bielawska and Y. A. H a n n u n , Methods" Enzyrnol. 311 [43] 1999 (this volume). 2a H. E. Carter, D. S. Galanos, and J. Fujino, Can. J. Biochem. Physiol. 34, 320 (1956). 2b W. Stoffel and G. Sticht, Hoppe-Seyler's Z. Physiol. Chem. 348, 1561 (1967). 2c E. L. ElM, S. H. Wilen, and L. N. Mander, "Stereochemistry of Organic C o m p o u n d s . " Wiley, New York, 1994.
METHODS IN ENZYMOLOGY,VOL. 311
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